Prospective evaluation of NGS-based sequencing in epilepsy patients: results of seven NASGE-associated diagnostic laboratories.

Background: Epilepsy is one of the most common and disabling neurological disorders. It is highly prevalent in children with neurodevelopmental delay and syndromic diseases. However, epilepsy can also be the only disease-determining symptom. The exact molecular diagnosis is essential to determine prognosis, comorbidity, and probability of recurrence, and to inform therapeutic decisions. Methods and materials: Here, we describe a prospective cohort study of patients with epilepsy evaluated in seven diagnostic outpatient centers in Germany. Over a period of 2 months, 07/2022 through 08/2022, 304 patients (317 returned result) with seizure-related human phenotype ontology (HPO) were analyzed. Evaluated data included molecular results, phenotype (syndromic and non-syndromic), and sequencing methods. Results: Single exome sequencing (SE) was applied in half of all patients, followed by panel (P) testing (36%) and trio exome sequencing (TE) (14%). Overall, a pathogenic variant (PV) (ACMG cl. 4/5) was identified in 22%; furthermore, a significant number of patients (12%) carried a reported clinically meaningful variant of unknown significance (VUS). The average diagnostic yield in patients ≤ 12 y was higher compared to patients >12 y cf. Figure 2B vs. Figure 3B. This effect was more pronounced in cases, where TE was applied in patients ≤ 12 vs. >12 y [PV (PV + VUS): patients ≤ 12 y: 35% (47%), patients > 12 y: 20% (40%)]. The highest diagnostic yield was achieved by TE in syndromic patients within the age group ≤ 12 y (ACMG classes 4/5 40%). In addition, TE vs. SE had a tendency to result in less VUS in patients ≤ 12 y [SE: 19% (22/117) VUS; TE: 17% (6/36) VUS] but not in patients >12 y [SE: 19% (8/42) VUS; TE: 20% (2/10) VUS]. Finally, diagnostic findings in patients with syndromic vs. non-syndromic symptoms revealed a significant overlap of frequent causes of monogenic epilepsies, including SCN1A, CACNA1A, and SETD1B, confirming the heterogeneity of the associated conditions. Conclusion: In patients with seizures-regardless of the detailed phenotype-a monogenic cause can be frequently identified, often implying a possible change in therapeutic action (36.7% (37/109) of PV/VUS variants); this justifies early and broad application of genetic testing. Our data suggest that the diagnostic yield is highest in exome or trio-exome-based testing, resulting in a molecular diagnosis within 3 weeks, with profound implications for therapeutic strategies and for counseling families and patients regarding prognosis and recurrence risk.

Clinical Experience with Noninvasive Prenatal Testing in Twin Pregnancy Samples at a Single Center in Germany.

In this study we wanted to determine the performance of a paired-end sequencing-based noninvasive prenatal testing (NIPT) assay in the detection of common fetal trisomies in twin pregnancy samples. Samples from patients with a twin pregnancy were collected from at least 10 weeks of gestation and analyzed at a single prenatal center in Germany. Results of Anomaly Detected (i.e., high risk) or No Anomaly Detected (i.e., low risk) for trisomy 21, trisomy 18, or trisomy 13 were reported. Follow-up confirmatory outcomes were requested for all cases. A total of 1,658 patients with twin pregnancies submitted samples during the study period; only two of these samples failed resulting in a low failure rate of 0.12%. Of the remaining 1,656 cases, there were 1,625 (98.1%) low-risk and 31 (1.9%) high-risk NIPT samples in our cohort. Of these, follow-up information was available for 301 (18.5%) of the low-risk samples and 19 (61.3%) of the high-risk samples. All of the low-risk cases with follow-up were determined to be true negatives giving an estimated negative predictive value of 100%. Seventeen of the 19 high-risk samples with follow-up were true positives, resulting in an overall positive predictive value of 89.5%. Sensitivities of > 99.9% were noted for both trisomy 21 and trisomy 18, with high specificities of ≥ 99.7% observed for all three trisomies. In conclusion, our study showed strong performance of the NIPT assay in the detection of common fetal trisomies in twin pregnancy samples, with high sensitivities, specificities, and positive predictive values observed based on known clinical outcomes along with a low failure rate.

Quality assurance within the context of genome diagnostics (a german perspective).

The rapid and dynamic implementation of Next-Generation Sequencing (NGS)-based assays has revolutionized genetic testing, and in the near future, nearly all molecular alterations of the human genome will be diagnosable via massive parallel sequencing. While this progress will further corroborate the central role of human genetics in the multidisciplinary management of patients with genetic disorders, it must be accompanied by quality assurance measures in order to allow the safe and optimal use of knowledge ascertained from genome diagnostics. To achieve this, several valuable tools and guidelines have been developed to support the quality of genome diagnostics. In this paper, authors with experience in diverse aspects of genomic analysis summarize the current status of quality assurance in genome diagnostics, with the aim of facilitating further standardization and quality improvement in one of the core competencies of the field.

Coordination Chemistry of Phosphate Groups in Systems Including Copper(II) Ions, Phosphoethanolamine and Pyrimidine Nucleotides.

The activity of phosphate groups of phosphoethanolamine and pyrimidine nucleotides (thymidine 5-monophosphate, cytidine 5-monophosphate and uridine 5'monophosphate) in the process of complexation metal ions in aqueous solution was studied. Using the potentiometric method with computer calculation of the data and spectroscopic methods such as UV-Vis, EPR, 13C and 31P NMR as well as FT-IR, the overall stability constants of the complexes as well as coordination modes were obtained. At lower pH, copper(II) ions are complexed only by phosphate groups, whereas the endocyclic nitrogen atom of nucleotides has been identified as a negative center interacting with the -NH3+ groups of phosphoethanolamine.

The Valence and Spin State Tuning of Iron(II/III) Porphyrazines with Bulky Pyrrolyl Periphery in Solution and Solid State.

Iron(III) porphyrazines containing peripheral 2,5-dimethyl-, 2-methyl-5-phenyl-, and 2,3,5-triphenyl-1H-pyrrol-1-yl substituents were synthesized and subjected to physicochemical characterization. This was accomplished by high-resolution mass spectrometry, nuclear magnetic resonance (as diamagnetic Fe(II) derivatives), HPLC purity analysis, and UV-Vis spectroscopy, accompanied by the solvation study in dichloromethane and pyridine. X-ray structure analysis was performed for a single crystal of the previously obtained 2,5-diphenyl-substituted derivative of porphyrazine complex (5d). The octahedral geometries of iron cation, present in the porphyrazine core, influenced the packing mode of molecules in the crystals. Mössbauer studies, performed for solid samples of iron porphyrazines, indicated that low-spin reduced iron states might occupy low- or high-symmetry binding sites. It was found that the hyperfine parameters and the subsequent contribution of the iron cations depend on the number of phenyl groups surrounding the pyrrolyl moiety. For iron(II) porphyrazine 2,3,5-triphenylpyrrol-1-yl substituents (5b), a high-spin ferrous state fraction was observed. Temperature-dependent measurements showed that the freed rotation of the peripheral porphyrazine ligands and the increased flexibility of the macrocycle ring result in the Fe2+ ion being stabilized in a diamagnetic state at a binding site of high symmetry at room temperature in the solid state. This process is most probably stimulated by the range of collective motions of the polymeric ribbons consisting of iron(II) porphyrazines observed in the X-ray.

Novel Short PEG Chain-Substituted Porphyrins: Synthesis, Photochemistry, and In Vitro Photodynamic Activity against Cancer Cells.

This work presents the synthesis and characterization of metal-free, zinc (II), and cobalt (II) porphyrins substituted with short PEG chains. The synthesized compounds were characterized by UV-Vis, 1H and 13C NMR spectroscopy, and MALDI-TOF mass spectrometry. The origin of the absorption bands for tested compounds in the UV-Vis range was determined using a computational model based on the electron density functional theory (DFT) and its time-dependent variant (TD-DFT). The photosensitizing activity was evaluated by measuring the ability to generate singlet oxygen (ΦΔ), which reached values up to 0.54. The photodynamic activity was tested using bladder (5637), prostate (LNCaP), and melanoma (A375) cancer cell lines. In vitro experiments clearly showed the structure-activity relationship regarding types of substituents, their positions in the phenyl ring, and the variety of central metal ions on the porphyrin core. Notably, the metal-free derivative 3 and its zinc derivative 6 exerted strong cytotoxic activity toward 5637 cells, with IC50 values of 8 and 15 nM, respectively. None of the tested compounds induced a cytotoxic effect without irradiation. In conclusion, these results highlight the potential value of the tested compounds for PDT application.

Sulfanyl Porphyrazines with Morpholinylethyl Periphery-Synthesis, Electrochemistry, and Photocatalytic Studies after Deposition on Titanium(IV) Oxide P25 Nanoparticles.

The syntheses, spectral UV-Vis, NMR, and electrochemical as well as photocatalytic properties of novel magnesium(II) and zinc(II) symmetrical sulfanyl porphyrazines with 2-(morpholin-4-yl)ethylsulfanyl peripheral substituents are presented. Both porphyrazine derivatives were synthesized in cyclotetramerization reactions and subsequently embedded on the surface of commercially available P25 titanium(IV) oxide nanoparticles. The obtained macrocyclic compounds were broadly characterized by ESI MS spectrometry, 1D and 2D NMR techniques, UV-Vis spectroscopy, and subjected to electrochemical studies. Both hybrid materials, consisting of porphyrazine derivatives embedded on the titanium(IV) oxide nanoparticles' surface, were characterized in terms of particle size and distribution. Next, they were subjected to photocatalytic studies with 1,3-diphenylisobenzofuran, a known singlet oxygen quencher. The applicability of the obtained hybrid material consisting of titanium(IV) oxide P25 nanoparticles and magnesium(II) porphyrazine derivative was assessed in photocatalytic studies with selected active pharmaceutical ingredients, such as diclofenac sodium salt and ibuprofen.

Analysis of cell-free DNA in a consecutive series of 13,607 routine cases for the detection of fetal chromosomal aneuploidies in a single center in Germany.

PURPOSE: Noninvasive prenatal testing (NIPT) is a highly sensitive and specific method for detection of fetal chromosomal aneuploidies from maternal plasma. The objective of this study was to determine the performance of a new paired-end sequencing-based NIPT assay in 13,607 pregnancies from a single center in Germany. METHODS: Samples from 13,607 pregnant women who previously underwent NIPT were analyzed using VeriSeq NIPT Solution v2 assay for presence of common fetal trisomies and monosomy X. Follow-up to determine clinical truth was carried out. RESULTS: Of the 13,607 cases, 13,509 received a NIPT call resulting in a low study failure rate of 0.72%. There were 188 (1.4%) high-risk calls: 117 trisomy 21, 34 trisomy 18, 23 trisomy 13, one trisomy 21 + 13, and 13 monosomy X. High sensitivities and specificities of ≥ 98.89% were reported for all four aneuploidy conditions. Of the high-risk cases, clinical follow-up data were available for 77.1% (145/188). Clinical follow-up of high-risk calls revealed an overall positive predictive value of 84.8% (potential range 65.4-88.3%). NIPT results were provided for samples across a range of fetal fractions, down to 2% fetal fraction. CONCLUSION: The VeriSeq NIPT Solution v2 assay detected fetal chromosomal aneuploidies across a range of fetal fractions with high sensitivities and specificities observed based on known clinical outcomes, a high overall PPV, and a low failure rate.

In vitro photodynamic activity of lipid vesicles with zinc phthalocyanine derivative against Enterococcus faecalis.

Zinc(II) phthalocyanine bearing eight non-peripheral 2-propoxy substituents was subjected to physicochemical study and, after incorporation in lipid vesicles, assessed as a potential photosensitizer for antibacterial photodynamic therapy. The phthalocyanine derivative obtained in the macrocyclization reaction was characterized by MS and NMR techniques. Moreover, its chemical purity was confirmed by HPLC analysis. X-ray structural analysis revealed that overcrowding of the phthalocyanine derivative leads to a strong out-of-plane distortion of the π-system of the macrocycle core. In the UV-Vis absorption spectra of zinc(II) phthalocyanine two characteristic bands were found: the Soret (300-450 nm) and the Q band (600-800 nm). Photophysical properties of mono- and diprotonated forms of phthalocyanine derivative were studied with time-resolved fluorescence spectroscopy. Its tri- and tetraprotonated forms could not be obtained, because compound decomposes in higher acid concentrations. The presented zinc(II) phthalocyanine showed values of singlet oxygen generation ΦΔ = 0.18 and 0.16, the quantum yield of the photodecomposition ΦP = 3.06∙10-4 and 1.23∙10-5 and the quantum yield of fluorescence ΦFL = 0.005 and 0.004, designated in DMF and DMSO, respectively. For biological studies, phthalocyanine has been incorporated into modified liposome vesicles containing ethanol. In vitro bacteria photoinactivation study revealed no activity against Escherichia coli and 5.7 log reduction of the Enterococcus faecalis growth.

Design, synthesis, and biological evaluation of novel combretastatin A-4 thio derivatives as microtubule targeting agents.

A series of novel combretastatin A-4 (CA-4) thio derivatives containing different molecular cores, namely α-phenylcinnamic acids (core 1), (Z)-stilbenes (core 2), 4,5-disubstituted oxazoles (core 3), and 4,5-disubstituted N-methylimidazoles (core 4), as cis-restricted analogues were designed and synthesized. They were selected with the use of a parallel virtual screening protocol including the generation of a virtual combinatorial library based on an elaborated synthesis protocol of CA-4 analogues. The selected compounds were evaluated for antiproliferative activity against a panel of six human cancer cell lines (A431, HeLa, MCF7, MDA-MB-231, A549 and SKOV) and two human non-cancer cell lines (HaCaT and CCD39Lu). Moreover, the effect of the test compounds on the inhibition of tubulin polymerization in vitro was estimated. In the series studied here, oxazole-bridged analogues exhibited the most potent antiproliferative activity. Compounds 23a, 23e, and 23i efficiently inhibited tubulin polymerization with IC50 values of 0.86, 1.05, and 0.85 µM, respectively. Thio derivative 23i, when compared to its oxygen analogue 23j, showed a 5-fold higher inhibitory impact on tubulin polymerization. Compounds 23e and 23i, which showed both best cytotoxic and antitubulin activity, were further studied in terms of their effect on cell cycle distribution and proapoptotic activity. Compound 23e induced a statistically significant block of the cell cycle at the G2/M phase in A431, HaCaT, HeLa, MCF-7, MDA-MB-231, and SKOV-3 cells to an extent comparable to that observed in CA-4. In HeLa and SKOV-3 cells incubated with 23i, a concentration-dependent block of the G2/M phase was observed. The proapoptotic effect of 23e and 23i in A431, HaCaT, MCF-7, MDA-MB-231, and SKOV-3 was demonstrated with ELISA assay and double staining with Annexin V-FITC/PI. The results indicated that compound 23e and 23i may serve as novel lead compounds in research on more effective anticancer agents.

Synthesis, biological evaluation and docking studies of trans-stilbene methylthio derivatives as cytochromes P450 family 1 inhibitors.

Cytochromes P450 family 1 (CYP1) are responsible for the metabolism of procarcinogens, for example polycyclic aromatic hydrocarbons and aromatic and heterocyclic amines. The inhibition of CYP1 activity is examined in terms of chemoprevention and cancer chemotherapy. We designed and synthesized a series of trans-stilbene derivatives possessing a combination of methoxy and methylthio functional groups attached in different positions to the trans-stilbene skeleton. We determined the effects of synthesized compounds on the activities of human recombinant CYP1A1, CYP1A2 and CYP1B1 and, to explain the variation of inhibitory potency of methoxystilbene derivatives and their methylthio analogues, we employed computational analysis. The compounds were docked to CYP1A1, CYP1A2 and CYP1B1 binding sites with the use of Accelrys Discovery Studio 4.0 by the CDOCKER procedure. For CYP1A2 and CYP1B1, values of scoring functions correlated well with inhibitory potency of stilbene derivatives. All compounds were relatively poor inhibitors of CYP1A2 that possess the most narrow and flat enzyme cavity among CYP1s. For the most active CYP1A1 inhibitor, 2-methoxy-4'-methylthio-trans-stilbene, a high number of molecular interactions was observed, although the interaction energies were not distinctive.

Antimicrobial and anticancer photodynamic activity of a phthalocyanine photosensitizer with N-methyl morpholiniumethoxy substituents in non-peripheral positions.

Photodynamic therapy involves the use of a photosensitizer that is irradiated with visible light in the presence of oxygen, resulting in the formation of reactive oxygen species. A novel phthalocyanine derivative, the quaternary iodide salt of magnesium(II) phthalocyanine with N-methyl morpholiniumethoxy substituents, was synthesized, and characterized. The techniques used included mass spectrometry (MALDI TOF), UV-vis, NMR spectroscopy, and photocytotoxicity against bacteria, fungi and cancer cells. The phthalocyanine derivative possessed typical characteristics of compounds of the phthalocyanine family but the effect of quaternization was observed on the optical properties, especially in terms of absorption efficiency. The results of the photodynamic antimicrobial effect study demonstrated that cationic phthalocyanine possesses excellent photodynamic activity against planktonic cells of both Gram-positive and Gram-negative bacteria. The bactericidal effect was dose-dependent and all bacterial strains tested were killed to a significant degree by irradiated phthalocyanine at a concentration of 1×10-4M. There were no significant differences in the susceptibility of Gram-positive and Gram-negative bacteria to the applied photosensitizer. The photosensitivity of bacteria in the biofilm was lower than that in planktonic form. No correlation was found between the degree of biofilm formation and susceptibility to antimicrobial photodynamic inactivation. The anticancer activity of the novel phthalocyanine derivative was tested using A549 adenocarcinomic alveolar basal epithelial cells and the human oral squamous cell carcinoma cells derived from tongue (HSC3) or buccal mucosa (H413). No significant decrease in cell viability was observed under different conditions or with different formulations of the compound.

Phthalocyanine derivatives possessing 2-(morpholin-4-yl)ethoxy groups as potential agents for photodynamic therapy.

Three 2-(morpholin-4-yl)ethoxy substituted phthalocyanines were synthesized and characterized. Phthalocyanine derivatives revealed moderate to high quantum yields of singlet oxygen production depending on the solvent applied (e.g., in DMF ranging from 0.25 to 0.53). Their photosensitizing potential for photodynamic therapy was investigated in an in vitro model using cancer cell lines. Biological test results were found particularly encouraging for the zinc(II) phthalocyanine derivative possessing two 2-(morpholin-4-yl)ethoxy substituents in nonperipheral positions. Cells irradiated for 20 min at 2 mW/cm(2) revealed the lowest IC50 value at 0.25 µM for prostate cell line (PC3), whereas 1.47 µM was observed for human malignant melanoma (A375) cells. The cytotoxic activity in nonirradiated cells of novel phthalocyanine was found to be very low. Moreover, the cellular uptake, localization, cell cycle, apoptosis through an ELISA assay, and immunochemistry method were investigated in LNCaP cells. Our results showed that the tested photosensitizer possesses very interesting biological activity, depending on experimental conditions.